Nitric oxide-releasing amidine—and enamine-derived diazeniumdiolates, compositions and uses thereof and method of making same

ABSTRACT

The present invention relates to nitric oxide-releasing amidine- and enamine-derived diazeniumdiolates, compositions comprising such compounds, methods of using such compounds and compositions, and to a method for the preparation of nitric oxide-releasing amidine- and enamine-derived diazeniumdiolates via the direct reaction of nitric oxide with amidines and enamines, and to a method of converting amines into such compounds.

PRIORITY

This patent application is a divisional of U.S. patent application Ser.No. 09/825,073, filed on Apr. 3, 2001, now U.S. Pat. No. 6,511,991,which is a divisional of U.S. patent application Ser. No. 09/446,653,filed on Mar. 30, 2000, now U.S. Pat. No. 6,232,336, which claims thebenefit of PCT/US98/13723, filed on Jul. 1, 1998, which claims thebenefit of U.S. patent application Ser. No. 60/051,690, filed on Jul. 3,1997.

FIELD OF THE INVENTION

The present invention relates to nitric oxide-releasing amidine- andenamine-derived diazeniumdiolates, to compositions comprising suchcompounds, to methods of using such compounds and compositions, to amethod for the preparation of nitric oxide-releasing amidine- andenamine-derived diazeniumdiolates via the direct reaction of nitricoxide with amidines and enamines, and to a method of converting amine'sinto such compounds.

BACKGROUND OF THE INVENTION

Nitric oxide (NO) has been implicated as part of a cascade ofinteracting agents involved in a wide variety of bioregulatoryprocesses, including the physiological control of blood pressure,macrophage-induced cytostasis and cytotoxicity, and neurotransmission(Moncada et al., “Nitric Oxide from L-Arginine: A Bioregulatory System,”Excerpta Medica, International Congress Series 897, Elsevier SciencePublishers B. V.: Amsterdam (1990); Marletta et al., Biofactors 2:219-225 (1990); Ignarro, Hypertension (Dallas) 16: 477-483 (1990);Kerwin et al., J. Med. Chem. 38: 4343-4362 (1995); and Anggard, Lancet343: 1199-1206 (1994)). Given that NO plays a role in such a widevariety of bioregulatory processes, great effort has been expended todevelop compounds capable of releasing NO. Some of these compounds arecapable of releasing NO spontaneously, e.g., by hydrolysis in aqueousmedia, whereas others are capable of releasing NO upon being metabolized(Lefer et al., Drugs Future 19: 665-672 (1994)).

Keefer et al. (U.S. Pat. Nos. 4,954,526; 5,039,705; 5,155,137; 5,208,233and 5,405,919 and related patents and patent applications, all of whichare incorporated herein by reference) disclose, among others, the use ofcertain nucleophile/nitric oxide adducts as NO-releasing agents, i.e.,

in which the nucleophilic residue (Nuc) is a primary amine, a secondaryamine or a polyamine. Although such adducts offer many advantages overother currently available nitric oxide-releasing compounds, onedisadvantage presented by the use of such adducts as pharmaceuticalagents is the potential risk of release of nitrosamines, which arecarcinogenic, upon decomposition and release of NO. Another disadvantageof the adducts of primary amines is that they can be unstable even assolids due to a tendency to form traces of potentially explosivediazotates.

Several types of compounds of the general structure

have been known for many years. Traube (Liebigs Ann. Chem. 300: 81-123(1898)) reported the preparation of a number of such compounds and notedthat treatment of the compounds with acid produced a “brown gas.”Although brown gas suggests the release of NO, given that a brown gasalso may be produced in the disproportionation of nitrite, the releaseof brown gas by the compounds prepared by Traube is not, in and ofitself, evidence of NO release. Compounds of the structural typereported by Traube are known to require harsh treatment with mineralacids to release any gas which is, of course, incompatible with abiological utility.

Another compound, which has the structure

and which has been named cupferron, has been shown by Kubrina et al.,Izvestia Akademii Nauk SSSR Seriia Biologicheskaia 6: 844-850 (1988)) togenerate NO in vivo. In addition, the antibiotics alanosine(C(O)(OH)CH(NH₂)CH₂N(O)═NOH) and dopastin(CH₃CH═CHC(O)NHCH₂CH(i-propyl)-N(O)═NOH), as well as cupferron, havebeen shown to release NO in vivo by enzymatic oxidation (Alston et al.,J. Biol. Chem. 260: 4069-4074 (1985)).

More recently, Keefer et al., in U.S. Pat. No. 5,212,204, have broadlydescribed that an organic moiety may be linked via carbon to the N₂O₂ ⁻group. This patent does not disclose an amidine or enamine structure asthe nucleophile, nor does it teach the nature of the structuralcharacteristics that an organic moiety must possess to cause theresulting N₂O₂ ⁻ group to be a nitric oxide donor.

Some N₂O₂ ⁻-containing compounds have been disclosed to be useful ascuring agents in rubber manufacture, antiknock additives for gasoline,indicator dyes, explosives, corrosion inhibitors and fungicides (Danziget al., U.S. Pat. No. 3,309,373; Wiersdorff et al., Chem Abstracts 77:48034f (1972); Massengale, U.S. Pat. No. 2,635,978; and Metzger et al.,U.S. Pat. No. 2,954,314). However, the mechanism of the reported actionof these compounds was not described.

In this regard, a recent study of the N₂O₂ ⁻ group (Taylor et al., J.Org. Chem. 60: 435-444 (1995)) proposed a mechanism for the observed NOrelease. The proposed mechanism was based on quantum mechanicalcalculations which showed protonation at the terminal oxygen to be mostfavored thermodynamically in the case of N bound N₂O₂ ⁻.

None of the above disclosures, however, mention anything about therelease of nitroxyl (HNO, which, at the physiological pH of 7.4, existsas NO⁻) by this functional group. Recent results suggest that, undercertain conditions, many classes of “NO donors” may release some NO⁻(see the discussions for nitrosothiols and diazeniumdiolates as well asthe table of NO donors in Feelisch et al., Donors of Nitrogen Oxides, InMethods in Nitric Oxide Research, M. Feelisch and J. S. Stamler, Eds.,Ch. 7, pp. 71-115, John Wiley and Sons, New York (1996)).

To date, there are three compounds used to generate HNO in solution. Onecompound, Angeli's salt, which is the standard HNO source (Fukuto etal., J. Pharm. Exp. Ther. 263: 546-551 (1992)), is, of course, aninorganic salt. The other two compounds, acetylated Piloty's acid (Smithet al., J. Amer. Chem. Soc. 82: 5731-5740 (1960)) and benzoylatedhydroxycyanamide (Lee et al., J. Med. Chem. 35 3648-3652 (1992)) arepromising inhibitors of aldehyde dehydrogenase. However, even in thesecompounds, there is debate as to whether the observed physiologicaleffects are attributed to NO, or to NO⁻. For example, Piloty's acid hasbeen shown to release NO oxidatively under physiological conditions(Zamora et al., Biochem. J. 312: 333-339 (1995)).

Reports that superoxide dismutase can prolong the effects of NO via itsreversible reduction to NO⁻ (Murphy et al., PNAS USA 88: 10860-10864(1991)) and that NO⁻, itself, exhibits potent activity as a vasodilator(Fukuto et al., J. Pharm. Exp. Ther. 263: 546-551 (1992)) and as aninhibitor of aldehyde dehydrogenase (Lee et al., J. Med. Chem. 35:3648-3652 (1992)) suggest that compounds, which release either NO or NO⁻or mixtures of the two, are potentially useful pharmaceutical agents andmay even offer advantages over compounds that just release NO.

Despite the extensive literature available on NO and nitricoxide-releasing compounds, there remains a need for stable nitricoxide-releasing compounds in which the nitric oxide-releasing group N₂O₂⁻ is bonded directly to a carbon atom and which can be prepared fromcompounds that do not include a nitrogen atom suitable for conversion toa diazeniumdiolate.

Accordingly, it is an object of the present invention to provide achemical structural framework having an atomic and electronicarrangement such that an N₂O₂ ⁻ functional group attached thereto willserve as a spontaneous NO and/or NO⁻ donor. It is a further object ofthe present invention to provide a method for producing novel NO and/orNO⁻-releasing diazeniumdiolates in which the N₂O₂ ⁻ group is bound to acarbon atom. Another object of the present invention is to provide NO-and/or NO⁻-releasing derivatives of amidines and enamines. A relatedobject of the present invention is to provide NO- and/or NO⁻-releasingderivatives of known pharmaceutical agents. A more specific object is toprovide NO- and/or NO⁻-releasing derivatives of known pharmaceuticalagents whose nitrogen atoms do not provide suitable N-diazeniumdiolatesas nitric oxide donors. Yet another object of the present invention isto provide compositions comprising NO- and/or NO⁻-releasing derivativesof amidines and enamines. A further object of the present invention isto provide methods of using NO- and/or NO⁻-releasing derivatives ofamidine and enamine compounds, and compositions thereof. These and otherobjects of the present invention, as well as additional inventivefeatures, will be apparent from the description of the inventionprovided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides NO- or NO⁻-releasing diazeniumdiolateswhich are derived from an enamine or an amidine and in which the N₂O₂ ⁻functional group is bonded to a carbon atom. The present invention alsoprovides compositions comprising such diazeniumdiolate compounds, andmethods of using such compounds and compositions. The present inventionfurther provides a method of producing an NO- or NO⁻-releasing enamine-or amidine-derived diazeniumdiolate. Additionally, the present inventionprovides a method for the preparation of an NO- and/or NO⁻-releasingamidine derivative from an existing amino compound. The method comprisesreaction of the amino compound with an acetamidating reagent followed byreaction with nitric oxide gas.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one aspect of the invention, there is provided anovel class of nitric oxide-nucleophile adducts or diazeniumdiolateshaving an amidine- or enamine-derived chemical linkage in which the N₂O₂⁻ functionality is bound directly to a carbon atom of the linkage. Theamidine- or enamine-derived chemical linkage which includes the N₂O₂ ⁻functional group is represented by the schematic formula depicting thecharacteristic connectivity:

wherein

C²—C³ means either C²—C³ or C²═C³

m is 1 or 2

q is 0 or 1

p is 0 or 1

provided that

(1) C² is tetravalent, and bound to two or more of C¹, C³, N¹ and N²;

(2) when p=1, then q=0 and C²—C³ means C²—C³; or

(3) when p=0, and q=1, then C²—C³ means either (i) C²═C³ or (ii) C²—C³where C²—N¹ means C²═N¹;

(4) when C²—C³ means C²—C³ and q=1 and p=0

C²—N¹ and C²—N² means

It will be appreciated by those skilled in the art that due to thenature of the synthesis reaction employed as disclosed herein, thedouble bond in all cases would originally form as a C═N and thentautomerize if that is possible due to the presence of a C—H β to N¹.The double bond typically tautomerizes to the more thermodynamicallyfavored structure. However, less thermodynamically favored tautomers mayoccur and have been observed depending on conditions such as solvent orthe like. In compounds where there is no H in the β position to N¹ notautomerization occurs. Thus, the present invention contemplates allNO-releasing diazeniumdiolates which include an amidine- or anenamine-derived chemical linkage in which the N₂O₂ ⁻ functional group isbound to a carbon atom irrespective of the tautomer that isthermodynamically favored. The electron movement or tautomerization forthe enamines and for the amidines is the same conceptually, but in thecase of the enamines it is the lone pair of electrons associated withthe nitrogen atom which must be used in the reaction since there is no Hon the enamine nitrogen.

The amidine- and enamine-based diazeniumdiolates of the presentinvention are advantageous in several respects. These compounds are notexpected to decompose to carcinogenic nitrosamines. Thediazeniumdiolates of the present invention exhibit the full range ofwater solubility. Some of the diazeniumdiolates of the present inventionare thus particularly useful where water insolubility is desirable, suchas in stents, implants, prostheses and the like. Many diazeniumdiolatesof the present invention are characterized by long-term slow release ofNO and can be used in coatings or the like. Further, these compounds donot bleed out of the coating, even after the NO has been released. Thediazeniumdiolates of the present invention are very stable solids and insolution are more heat stable than the previously described nitrogenanalogs. Some can be recrystallized from boiling solvents withoutdecomposition.

In keeping with the invention, the amidine-derived diazeniumdiolates maybe further described in accordance with the following formulas:

wherein R¹-R⁵ can be a wide variety of substituents without departingfrom the scope of the present invention owing to the fact that anycompound which includes the characteristics of the chemical linkageidentified above is contemplated herein.

Thus, in the compounds of Formula I, II or III, R¹-R³ are independentlychosen from hydrogen, an unsubstituted or substituted C₁₋₁₂ straightchain alkyl, an unsubstituted or substituted C₃₋₁₂ branched chain alkyl,an unsubstituted or substituted C₃₋₁₂ straight chain olefinic, anunsubstituted or substituted C₃₋₁₂ branched chain olefinic, asubstituted or unsubstituted C₃₋₁₂ cycloalkyl, a C₃₋₈ heterocyclic ringbound through a carbon atom and in which the heteroatom is oxygen ornitrogen, a substituted or unsubstituted naphthyl, a substituted orunsubstituted tetrahydronaphthyl, a substituted or unsubstitutedoctrahydronaphthyl, benzyl or substituted benzyl, substituted with up tothree substituents, or a substituted or unsubstituted phenyl,substituted with up to three substituents.

In the compounds of Formula I, II or III, R⁴ and R⁵ are independentlyhydrogen, an unsubstituted or substituted C₁₋₁₂ straight chain alkyl, anunsubstituted or substituted C₃₋₁₂ branched chain alkyl, anunsubstituted or substituted C₃₋₁₂ straight chain olefinic, anunsubstituted or substituted C₃₋₁₂ branched chain olefinic, asubstituted or unsubstituted benzyl, a substituted or unsubstitutedphenyl, a substituted or unsubstituted piperazino, or a substituted orunsubstituted morpholino. R⁴ and R⁵ also can be amino, an unsubstitutedor substituted alkylamino, carboxyalkylamino, carboxydialkylamino, anunsubstituted or substituted tolyl, xylyl, anisyl, mesityl, nitro, anunsubstituted or substituted arylamino, an unsubstituted or substituteddialkylamino, an unsubstituted or substituted diarylamino, anunsubstituted or substituted acetyl, an unsubstituted or substitutedacetoxy, carboxy, an unsubstituted or substituted carboxyalkyl, such asan unsubstituted or substituted carboxymethyl or an unsubstituted orsubstituted carboxyethyl, an unsubstituted or substituted alkylcarbonyl,thiol, an unsubstituted or substituted alkylthio, an unsubstituted orsubstituted alkoxy, carboxamido, an unsubstituted or substitutedalkylcarboxamido, an unsubstituted or substituted dialkylcarboxamido, anunsubstituted or substituted phenoxy, an unsubstituted or substitutedbenzyloxy, an unsubstituted or substituted nitrophenyl, phenylcarbonyl,benzylcarbonyl, trialkylsilyl.

When any of the groups indicated above for R¹-R⁵ are identified as beingsubstituted, such as when the C₁₋₁₂ straight chain alkyl, the C₃₋₁₂branched chain alkyl, the C₃₋₁₂ straight chain olefinic, the C₃₋₁₂branched chain olefinic, the C₃₋₈ cycloalkyl, the benzyl, piperazino,morpholino, alkylamino, arylamino, acetyl, acetoxy, carboxy,carboxymethyl, alkoxy or the like are substituted, they can besubstituted with any moiety that does not destroy the NO-releasingcharacter of the compounds and which, preferably, is biologicallycompatible. Accordingly, substituents to the substituted R¹-R⁵ groupscan include hydroxy, alkoxy, acyloxy, halo or benzyl, acetyl, carboxyl,carboxyalkyl, such as carboxymethyl, carboxyethyl, carboxyalkylamido,carboxydialkylamido, carboxamido, amino, alkylamino, dialkylamino,alkylcarbonyl, arylamino, diarylamino, cyano, tolyl, xylyl, mesityl,anisyl, pyrrolidinyl, formyl, dioxane, thiol, alkylthiol, aryl,heteroaryl, such as pyran, pyrrole, furan, thiophene, thiazole,pyrazole, pyridine, or pyrimidine, phenoxy, benzyloxy, phenylcarbonyl,benzylcarbonyl, nitrophenyl trialkylsilyl, nitro, sulfonyl, nitrobenzyl,trialkylammonium, alkyl, cycloalkyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl or morpholinyl.

The substituents R¹, R², R³, R⁴ and R⁵, in various combinations, andtogether with the nitrogen atom or carbon atom to which they are bonded,can form unsubstituted or substituted cyclic or unsubstituted orsubstituted heterocyclic rings. The rings that are formed are fourmember rings or layers. For example, R¹ and R² together with thenitrogen atoms to which they are bonded can form a C₂₋₈ heterocyclicring. R¹ and R⁴ together with the nitrogen atom to which R¹ is bondedand with the carbon atom to which R⁴ is bonded can form a C₃-C₈heterocyclic ring. Similarly, R² and R³ can form a C₃₋₈ heterocyclicring with the nitrogen atom to which they are bonded. The heterocyclicring can also include up to one additional heteroatom, such as oxygen,nitrogen or sulfur.

The heterocyclic rings formed by the different combinations of R¹, R²,R³, R⁴ and R⁵ can be, for example, a piperazino, a morpholino, ahexamethyleneimino, an imidazolyl, a pyrrolidino, a piperidino or thelike. Likewise, R⁴ and R⁵ together with the carbon atom to which theyare bonded can form a C₃₋₈ cycloalkyl, or a heterocyclic such astetrahydrofuranyl, dioxanyl or the like. Further, R⁴ and R⁵ togetherwith the carbon atom to which they are bonded can form a1,4-benzodioxane, 1,3-benzodioxole, tetrahydronaphthlene,octahydronaphthalene, piperazine, morpholine, tetrahydroquinoline,tetrahydroquinoxaline, or tetrahydroisoquinoline.

Each of the cyclic or heterocyclic rings formed with R¹ and R², or R²and R³, or R¹ and R⁴, or R⁴ and R⁵ can be substituted with one or moresubstituents, including, by way of example, C₃₋₈ cycloalkyl, alkoxy,benzyl, fused benzene, phenyl, an alkoxy, acetyl, carboxyl,carboxymethyl, carboxyethyl, carboxamido, amino, alkyl amino,dialkylamino, pyrrolidine, dioxane, thiol or alkylthiol, or a heteroarylsuch as pyran, pyrrole, furan, thiophene, thiazole, pyrazole, pyridine,or pyrimidine.

The compounds of the present invention can be derived from existingpharmaceutical agents that contain the amidine group. For example, acompound of Formula III preferably is one in which R¹ and R² arehydrogen and R³ is the entire substituent attached to an amine of apharmaceutical agent such as, for example, tryptamine, serotonin,histamine, valcyclovir, adenosine, thyroxine, guanine, guanosine,ubenimex, glucosamine, mannosamine, mycosamine, sphingosine,thienamycin, penicillamine and rimantadine. Similarly, for example, thepresent invention provides a compound of Formula III, in which R¹ and R²are hydrogen and R³ is the entire substituent attached to an amine of anamino acid. The amino acid is preferably lysine, tryptophan orhydroxy-tryptophan.

The present invention also provides compounds of

wherein R¹-R⁶ can be a wide variety of substituents without departingfrom the scope of the present invention owing to the fact that anycompound which includes the characteristics of the chemical linkageidentified above is contemplated herein.

Thus, in the compounds of Formula IV and Formula V, R¹, R², R⁵ and R⁶are independently hydrogen, an unsubstituted or substituted C₁₋₁₂straight chain alkyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkyl, an unsubstituted or substituted C₃₋₁₂ straight chainolefinic, an unsubstituted or substituted C₃₋₁₂ branched chain olefinic,a substituted or unsubstituted benzyl, a substituted or unsubstitutedpiperazino, a substituted or unsubstituted morpholino, amino, anunsubstituted or substituted alkylamino, an unsubstituted or substitutedarylamino, an unsubstituted or substituted dialkylamino, anunsubstituted or substituted diarylamino, carboxyalkylamino,carboxydialkylamino, cyano, tolyl, xylyl, anisyl, mesityl, nitro, anunsubstituted or substituted acetyl, an unsubstituted or substitutedacetoxy, carboxy, an unsubstituted carboxyalkyl, such as anunsubstituted or substituted carboxymethyl, or an unsubstituted orsubstituted carboxyethyl, an unsubstituted or substituted alkylcarbonyl,thiol, an unsubstituted or substituted alkylthio, an unsubstituted orsubstituted alkoxy, carboxamido, an unsubstituted or substitutedalkylcarboxamido, or an unsubstituted or substituted dialkylcarboxamido.

In the compounds of Formula IV and V, R³ and R⁴ are independently chosenfrom hydrogen, an unsubstituted or substituted C₁₋₁₂ straight chainalkyl, an unsubstituted or substituted C₃₋₁₂ branched chain alkyl, anunsubstituted or substituted C₃₋₁₂ straight chain olefinic, anunsubstituted or substituted C₃₋₁₂ branched chain olefinic, asubstituted or unsubstituted C₃₋₈ cycloalkyl, a C₃₋₈ heterocyclic ringbound through a carbon atom and in which the heteroatom is oxygen ornitrogen, a substituted or unsubstituted naphthyl, a substituted orunsubstituted tetrahydronaphthyl, a substituted or unsubstitutedoctahydronaphthyl, benzyl or substituted benzyl, substituted with up tothree substituents, or a substituted or unsubstituted phenyl,substituted with up to three substituents. Such compounds areadvantageous because they are more “organic” than polyamines, such thatsimple aromatic enamines can be made to be water-insoluble, yet releaseNO, and to be heat-stable.

When any of the groups indicated above for R¹-R⁵ are identified as beingsubstituted, such as the C₁₋₁₂ straight chain alkyl, the C₃₋₁₂ branchedchain alkyl, the C₃₋₁₂ straight chain olefinic, the C₃₋₁₂ branched chainolefinic, the C₃₋₈ cycloalkyl, the benzyl, piperazino, morpholino,alkylamino, arylamino acetyl, acetoxy carboxy, carboxymethyl alkoxy orthe like, they can be substituted with any moiety that does not destroythe NO-releasing character of the compounds and which, preferably, isbiologically compatible. Accordingly, substituents to the substitutedR¹-R⁵ groups can include hydroxy, alkoxy, acyloxy, halo or benzyl,acetyl, carboxyl, carboxyalkyl, such as carboxymethyl, carboxyethyl,carboxyalkylamido, carboxydialkylamido, carboxamido, amino, alkyl amino,dialkylamino, alkylcarbonyl, arylamino, diarylamino, tolyl, xylyl,mesityl, anisyl, pyrrolidine, formyl, dioxane, thiol, alkylthiol, aryl,heteroaryl, such as pyran, pyrrole, furan, thiophene, thiazole,pyrazole, pyridine, or pyrimidine, phenoxy, benzyloxy, phenylcarbonyl,benzylcarbonyl, nitrophenyl trialkylsilyl, nitro.

The groups R¹-R⁶ of the compounds of Formula IV and Formula V in variouscombinations, and together with the nitrogen atom or carbon atom towhich they are bonded and intervening atoms, can form heterocyclicrings. For example, and not in limitation, a compound of Formula V inwhich R³ and R⁴, together with the nitrogen atom to which they arebonded, can form a C₃₋₈ heterocycle. The heterocycle can be furthersubstituted with a heteroatom. As another example, in the Formula Vcompound, R¹ and R⁶, together with the C═C—C through which they arebonded, can form a substituted or unsubstituted C₃₋₁₂ cycloalkyl.Similarly, for a compound of Formula IV, R² and R³, together with thenitrogen to which R³ is bonded, can form a C₃₋₈ heterocycle. Theheterocycle can be further substituted with a heteroatom, or an aromaticring, which can be substituted with a C₁₋₆ alkyl or a C₁₋₆ alkoxy. Also,R⁵ and R⁴ can form a C₃₋₈ heterocycle, which can also be substituted.

In Formulas IV and V, R³ and R⁴ together with the nitrogen atom to whichthey are bonded can form a C₃₋₈ heterocyclic ring or a C₃₋₈ substitutedheterocyclic ring or a C₃₋₈ unsubstituted or substituted heterocyclicring containing up to two additional heteroatoms selected from the groupO, S, N.

Also, R⁵ and R⁶ together with the carbon to which they are bonded canform a substituted or unsubstituted C₄₋₈ cycloalkyl.

With respect to the compounds of Formulas I, II and III, R¹-R⁵ can beselected such that they represent the substituents attached to theamidine of nasal decongestants and α-adrenergic antagonists such astetrahydrozoline, idazoxan, phentolamine, xylometazoline and the like.

In accordance with another aspect of the invention, there is provided amethod for the preparation of the amidine- and enamine-derivedNO-releasing compounds described herein. In one embodiment, the methodcomprises reacting an amidine, preferably an amidine of Formula Ia, IIaor IIIa, with gaseous NO in acetonitrile or a similar solvent to producean N₂O₂ ⁻-containing compound. R¹ and R⁴ together with the nitrogen atomto which R¹ is bonded and with the carbon atom to which R⁴ is bonded canform a C₃-C₈ heterocyclic ring.

The solvent is preferably chosen so that the is starting amidine orenamine is soluble whereas the resulting NO₂ ⁻-containing product isinsoluble and so precipitates as it forms in order to drive the reactionto completion. Anhydrous and neutral solvents such as acetonitrile,tetrahydrofuran, dioxane and ether are preferred because they do notcause hydrolysis of the water-sensitive amidines and enamines. However,it is anticipated that low yields of the desired products can also formin partly aqueous and/or basic solvents such as NaOMe in methanol or wettetrahydrofuran among others, and such solvents may also be used.

The resulting compound in accordance with the method of the inventioncontains either one or two N₂O₂ ⁻ functional groups depending upon thestructure of the amidine reactant, as, for example, shown below.

Methods for the preparation of the amidines, such as those of FormulasIa, IIa and IIIa, are well known and have been reviewed in two referencebooks, Gautier et al., “Preparation and Synthetic Uses of Amidines,”Chapter 7 in The Chemistry of Amidines and Imidates, Editor: Patai, pp.283-348, Wiley, 1975, and Boyd, “Recent Advances in the Synthesis ofAmidines,” Chapter 7 in The Chemistry of Amidines and Imidates, Volume2, Editors: Patai and Rappoport, pp. 339-3.67, Wiley, 1991. Thesemethods can be used by those skilled in the art to prepare a widevariety of amidines which can then be made into NO-releasingdiazeniumdiolates in accordance with the invention.

By way of example and not in limitation, the preparation of anNO-releasing amidine-derived diazeniumdiolate can be illustrated by thereaction of 2-methyl-2-imidazoline with NO as follows:

Although the initial reaction products are the amidinium salts (eitherintramolecular or intermolecular), standard metathesis reactions can beemployed to change the cation to any pharmaceutically acceptable ion.This is illustrated above by the reaction involving sodium methoxide inmethanol, which produces the disodium salt. Also, by varying thesynthesis procedures, the intramolecular or intermolecular salt or amixture thereof can be obtained; the reaction of 2-methyl-2-imidazolinewith NO in NaOMe/MeOH to directly form the sodium salt is an example ofsuch a reaction.

While applicants do not wish to be bound to any particular theory, theabove reactions are believed to be explained by the reaction of NO withthe little exploited enediamine tautomers of the amidines. Theenediamine tautomers are known to exist in solution and were firstproposed to explain deuterium exchange in NMR solutions as follows(Isagulyants et al., Zh. Prikl. Khim. 41: 1585-1590 (1968); also, inChem. Abstracts 70: 11629h (1969)):

Accordingly, while not being bound to any particular theory, it isbelieved that the reaction of the above undeuterated compound with an NOdimer is as follows:

The reaction is believed to stop at this stage due to steric hindranceand/or precipitation of the product from solution.

When either the amidinium or sodium salt of the NO-releasingdiazeniumdiolate derived from 2-methyl-2-imidazoline was dissolved inwater and acidified, a voluminous gas evolution resulted and thesolution turned blue in color and remained so for many hours after gasevolution had ceased. When the experiment was repeated at pH 7.4, theevolving gas was identified as a mixture of 2 parts NO (determined bychemiluminescence) and 1 part N₂O (determined by gas chromatography).Nitrous oxide (N₂O), being the end product of HNO dimerization anddehydration, provided a measure of HNO production via the equation(Nagasawa et al., J. Med. Chem. 33: 3122-3124 (1990)):

Again, while not wishing to be bound to any particular theory, it isbelieved that the partial mechanistic explanation for these observationsis as follows:

The last step in this mechanism is not well understood but has precedentin the known release of NO by FK409 and closely related compounds whichare used as standard sources of NO (Kita et al., Eur. J. Pharmacol. 257:123-130 (1994)). Although this mechanism is one explanation for theobserved NO and N₂O release, it is a very incomplete representation ofwhat actually happens to any given compound in aqueous solution.Specifically, amidines are known to be subject to hydrolysis at ratesthat range from very slow, such as for 2-methyl-2-imidazoline (Haake etal., J. Org. Chem. 35: 4063-4067 (1970))

to very fast for acetamidine (Davies et al, Chem. Ind. (London): 628(1958)).

Thus, at any intermediate stage of HNO or NO release, the amidino groupcould hydrolyze and no further gas would be generated. A compound inwhich the amidine hydrolyzes rapidly would release much HNO but verylittle NO, whereas a compound in which the amidine hydrolyzes slowlywould have time for NO release, which is the last step, and would thusrelease a larger mount of NO. In this regard, compounds of Formula I (asset forth above) cannot be hydrolyzed by the above mechanism. It isbelieved that these mono-N₂O₂ ⁻ derivatives break down via two competingpathways, one of which appears to be simple reversal of the synthesisstep to release NO, while the other may be a single scission to yieldone molecule of HNO and a mono-C-nitroso compound. Since the amidinotautomers cannot come into conjugation with this nitroso group, it doesnot serve as a source of NO, and since hydrolysis of the amidinecompetes with the first pathway, compounds derived from amidines offormula I release only small amounts of NO, but over a long period oftime. In such cases, the reaction of an amidine with NO results in asterically hindered compound of formula I, which is apparently inclinedto break apart differently than previously reported, less hindered N₂O₂⁻ compounds.

In another embodiment of the present inventive method, an enamine,preferably an enamine of Formula IV or V, is reacted with NO to producean N₂O₂ ⁻ containing compound. Enamines are prepared from an equimolarmixture of an aldehyde or ketone and a secondary amine via dehydrationas follows.

Methods for preparing enamines and lengthy discussions of theirproperties are readily available to synthetic chemists (see, e.g.,Hickmott, Tetrahedron 38: 1975-2050 (1982); Hickmott, Tetrahedron 38:3363-3446 (1982); Cook, Enamines: Synthesis, Structure and Reactions,Marcel Dekker, New York (1988); and Szmuszkovicz, Enamines, Vol. 4 ofAdv. in Org. Chem. Methods and Results, Wiley Interscience, New York(1963)). Although literally thousands of carbonyl compounds are used inthis reaction, the amines are usually limited to a select few, such asdimethylamine, diethylamine, piperidine, pyrrolidine, morpholine, andN-methylaniline.

Unlike the amidine-derived compounds, the enamine-deriveddiazeniumdiolates do not appear to release any NO⁻ or N₂O. Rather, theyrelease small amounts of NO over prolonged periods of time (e.g., 1 weekin phosphate-buffered saline). As with amidine-derived compounds, themechanism of NO release is complicated by a competing hydrolysismechanism as set forth below.

It will be appreciated by those of ordinary skill in the art that eitherthe amidine-derived or enamine-derived diazeniumdiolates in accordancewith the present invention can be formed as a salt, and preferably, abiologically acceptable salt. Accordingly, the counterion is preferablyany biologically acceptable acceptable counterion. Such counterions caninclude, but are not limited to, sodium ion, potassium ion, quaternaryammonium ions, and the like.

Also provided by the present invention is a method of producing a nitricoxide-releasing compound from a compound containing a primary amineand/or a secondary amine. The method comprises (a) treating the compoundcontaining a primary amine and/or a secondary amine with anacetamidating agent, by which is meant an organic chemical reagentcapable of transferring the CH₃C (═NH)⁻ group from itself to anothermolecule. Such reagents are generally acetimidates, for example, ethylacetimidate, or thioimidates, for example, benzyl thioacetimidate. Thepreferred reagent for use in the context of this method is thatdescribed in Shearer et al., Tetrahedron Letters 38(2): 179-182 (1997),so as to form an acetamidine derivative of the compound containing theprimary amine and/or secondary amine, and (b) treating the acetamidinederivative with nitric oxide gas to form an amidine-deriveddiazeniumdiolate. This method in accordance with the invention providesa method for preparing an amidine-based diazeniumdiolate in which theNO-releasing N₂O₂ ⁻ functional group is bound to a carbon atom ratherthan to the original primary or secondary amine. In this way, manyprimary and secondary amine-containing drugs can be subjected to theacetamidating reagent to produce the amidine which can then be convertedto the diazeniumdiolate. This is advantageous particularly in the caseof primary amines where the N—N₂O₂ ⁻ functionality is not very stable.

As is well known in the art, nitric oxide and compounds comprising N₂O₂⁻ functional groups can have a wide range of utilities, in part becauseof the multifaceted role of nitric oxide in bioregulatory processes.Accordingly, the present invention also provides a composition,including a pharmaceutical composition, comprising a present inventivediazeniumdiolate. Preferably, the pharmaceutical compositionadditionally comprises a pharmaceutically acceptable carrier.

One skilled in the art will appreciate that suitable methods ofadministering a diazeniumdiolate composition of the present invention toan animal, such as a mammal, are available, and, although more than oneroute can be used to administer a particular composition, a particularroute can provide a more immediate and more effective reaction thananother route. Pharmaceutically acceptable carriers are also well-knownto those who are skilled in the art. The choice of carrier will bedetermined, in part, both by the particular composition and by theparticular method used to administer the composition. Accordingly, thereis a wide variety of suitable formulations of the pharmaceuticalcompositions of the present invention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the diazeniumdiolate dissolvedin diluents, such as water or saline, (b) capsules, sachets or tablets,each containing a predetermined amount of the active ingredient, assolids or granules, (c) suspensions in an appropriate liquid, and (d)suitable emulsions. Solutions may also be formulated using knownpreservatives for amidine-based nasal decongestants.

Tablet forms can include one or more of lactose, mannitol, corn starch,potato starch, microcrystalline cellulose, acacia, gelatin, colloidalsilicon dioxide, croscarmellose sodium, talc, magnesium stearate,stearic acid, and other excipients, colorants, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, such carriersas are known in the art.

The diazeniumdiolates of the present invention, alone or in combinationwith other suitable components, can be made into aerosol formulations tobe administered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration include aqueous andnon-aqueous solutions, isotonic sterile injection solutions, which cancontain anti-oxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The dose administered to an animal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the animal over a reasonable time frame. The dose will bedetermined by the strength of the particular compositions employed(taking into consideration, at least, the rate of NO evolution, theextent of NO evolution, and the bioactivity of any decompositionproducts derived, from the diazeniumdiolates) and the condition of theanimal, as well as the body weight of the animal to be treated. The sizeof the dose also will be determined by the existence, nature, and extentof any adverse side-effects that might accompany the administration of aparticular composition. A suitable dosage for internal administration is0.01 to 100 mg/kg per day. A preferred dosage is 0.01 to 35 mg/kg perday. A more preferred dosage is 0.05 to 5 mg/kg per day. A suitableconcentration of a enamine- or amidine-derived diazeniumdiolate inpharmaceutical compositions for topical administration is 0.05 to 15%(by weight). A preferred concentration is from 0.02 to 5%. A morepreferred concentration is from 0.1 to 3%.

In view of the above, the present invention provides methods of using anitric oxide-releasing amidine- or enamine-derived diazeniumdiolate. Inone embodiment, a method of treating an animal, such as a mammal, with abiological disorder treatable with nitric oxide, is provided. The methodcomprises administering to the animal, e.g., the mammal, in need thereofan amount of an enamine- or amidine-derived diazeniumdiolate sufficientto treat the biological disorder in the animal. In this embodiment,“biological disorder” can be any biological disorder, includinghypertension, restenosis, impotency, and a biological disorder due to agenetic defect or infection with an infectious agent, such as a virus,bacterium or parasite, as long as the disorder is treatable with nitricoxide.

With regard to the above, NO- and/or NO⁻-releasing compounds derivedfrom amidines are advantageous inasmuch as amidines are present in manyalready approved medicinal agents, e.g., tranquilizers, α-adrenergicantagonists, like phentolamine, and nasal decongestants. Specificexamples include tolazoline and diazoxide. Other examples ofamidine-containing compounds include methyl pyrimidine and 1,8-diaminooctahydronaphthalene.

In another embodiment of a method of use, a method is provided fortreating an animal, such as a mammal, for infection with, for example, avirus, a bacterium, or a parasite. The method comprises administering tothe animal, e.g., the mammal, an amount of a diazeniumdiolate sufficientto treat the infection in the animal.

In yet another embodiment, a method for treating an animal, such as amammal, for cancer is provided. The method comprises administering tothe animal, e.g., the mammal, an amount of diazeniumdiolate sufficientto prevent the growth or metastasis of the cancer in the animal or torender it more susceptible to radiation or chemotherapy.

In another embodiment, a method is provided for treating an inanimateobject for the presence of a potentially infectious virus, bacterium, orparasite. The method comprises contacting the inanimate object with anamount of a present inventive diazeniumdiolate sufficient to reduce thepresence of the potentially infectious virus, bacterium or parasite. By“potentially infectious” is meant the capability of infecting an animal,such as a mammal.

It is contemplated that the diazeniumdiolates derived from enamines andamidines in accordance with the present invention can be used to coatprostheses, stents, and medical implants, such as breast implants, priorto surgical introduction into the body as a means of reducing the riskof solid state carcinogenesis associated therewith, or as a means ofpreventing adhesion of platelets to the implants. Additionally, theprostheses and implants can be manufactured using an enamine- oramidine-derived diazeniumdiolate as an integral component of thestarting materials. Medical devices incorporating an enamine- oramidine-derived diazeniumdiolate provide an invaluable two-prongedapproach to the treatment of many biological disorders, providing usefulmedical structures that also advantageously provide local release of NO.

The diazeniumdiolates derived from enamines and amidines also haveutility in the in vitro study of NO biology.

EXAMPLES

The following examples further illustrate the present invention and, ofcourse, should not be construed as in any way limiting its scope.

All melting points were determined on a hot stage and are uncorrected.The ¹H NMR spectra were determined at 200 MHz with a Varian XL-200spectrometer and the ¹³C NMR spectra were obtained at 50 MHz using thesame instrument. The chemical shifts are expressed in δ values (ppm)relative to either tetramethylsilane or sodium3-(trimethylsilyl)propionate-d₄ as internal standards. Elementalanalyses were performed by Atlantic Microlabs, Inc. (Norcross, Ga.).

Except as noted here, all reagents and amines were obtained from AldrichChemical Company (Milwaukee, Wis.). Reaction solvents were Aldrichanhydrous grade but all others were reagent grade. Commercial gradenitric oxide was obtained from Matheson Gas Products and was used asreceived.

Reactions under pressure were conducted in standard glass hydrogenationbottles as previously described (Hrabie et al., J. Org. Chem. 58:1472-1476 (1993)). The general directions are repeated here forcompleteness.

Given that stainless steel (SS) is required for prolonged exposure to NOgas and amines degrade most types of stoppers and gaskets, a specializedreactor modeled after the standard Parr 3911 hydrogenation apparatus(Parr Instrument Co., Moline, Ill.) was constructed. The reservoir wasreplaced by a type 304 SS gas sampling cylinder equipped with SSfittings (available from any “valve and fitting” plumbing supplycompany). The valves were diaphragm-seal packless type (Aldrich), andthe pressure gauges were SS (Air Products). The usual Parr clamp andbottle system was employed but was connected to the gas reservoir via aTeflon tube and mounted to allow stirring with a magnetic stirrer.

All of the analytical data given were obtained using the products asisolated directly from the reaction mixtures.

Example 1

This example describes a generalized procedure for the preparation ofNO- and/or NO⁻-releasing compounds from amidines.

A solution of the appropriate amidine, which was obtained commercially(Aldrich) or synthesized in accordance with standard procedures, in thedesired solvent was placed in a standard Parr hydrogenation bottle.Nitrogen was passed through the apparatus and bubbled through thesolution for 5-10 min, the bottle was clamped, and NO gas was admittedto a pressure of 5 atm. The solution was stirred for the indicated timeat room temperature with addition of NO as needed during the first 5-6 hto maintain the reservoir pressure. Excess NO was then vented and N₂ wasbubbled through the resultant white slurry for 5 min. The product wasisolated by filtration, washed with the reaction solvent, then washedwith ether and dried in vacuo for several hours. All of the productswere amorphous, voluminous white powders, which were air-stable but werestored in a refrigerator.

Example 2

This example describes the preparation of 2-methyl-2-imidazolinetetrakis(nitric oxide)adduct and its sodium salt.

A solution of 2-methyl-2-imidazoline (lysidine, 5.0 g, 59.4 mmol) in 150ml acetonitrile was reacted with NO for 28 h as described above. Yield3.59 g (49%); m.p. 102-103° C. dec.; ¹H NMR (D₂O) δ1.92 (6H, s), 3.51(8H, s), 3.67 (4H, s); ¹³C NMR (D₂O) 24.8, 42.4, 42.5, 44.6, 51.3, 51.7,163.5, 177.2; UV (0.01 N NaOH) λ_(max) 260 nm,=13,600 M⁻¹cm⁻¹, 206 nm,ε=22,500. Anal. Calcd for C₁₂H₂₄N₁₀O₄: C, 38.71; H, 6.50; N, 37.61.Found: C, 38.92; H, 6.55; N, 37.62.

To prepare the disodium salt, 1.74 g of a 25% NaOMe in MeOH solution(Aldrich, 8.06 mmol) was diluted with 0.5 ml MeOH and to this was added1.5 g of the above diimidazolinium salt (8.06 mmol). The solid slowlydissolved and then re-precipitated. The slurry was diluted withacetonitrile, filtered and the solid dried in vacuo to afford a whitepowder. Yield 0.92 g (92%). m.p. >180° C. (chars); ¹H NMR (D₂O) δ2.7-2.8(2H, m), 3.3-3.4 (2H, m).

Example 3

This example describes the preparation of acetamidine tetrakis (nitricoxide) adduct.

A solution of acetamidine hydrochloride (7.0 g, 74.0 mmol) in 150 mlacetonitrile was treated with 16.93 ml of 25% NaOMe in MeOH (74.0 mmol)and the precipitated sodium chloride was removed by filtration. Theresulting solution was treated with NO for 16 h to yield a tan powder.Yield 5.95 g (82%); m.p. >150° C. (chars); ¹H NMR (D₂O) δ2.21 (s); ¹³CNMR (D₂O) 20.8, 51.8, 57.7, 164.6.

Example 4

This example describes the preparation of 2-iminopiperidine bis(nitricoxide)adduct.

A solution of 2-iminopiperidine hydrochloride (5.0 g, 37.2 mmol) in 200ml acetonitrile was treated with 8.5 ml of 25% NaOMe in MeOH (37.2 mmol)and 10 ml MeOH and the precipitated sodium chloride was removed byfiltration. The resulting solution was treated with NO for 23 h to yieldan off-white powder. Yield 4.5 g (95%); m.p. 110-112° C. (dec.); ¹H NMR(D₂O) δ1.8-1.9 (6H, m), 2.55-2.65 (2H, m), 2.85-2.95 (2H, m), 3.3-3.4(2H, m), 3.5-3.6 (2H, m); ¹³C NMR (D₂O) 19.0, 20.3, 23.0, 28.3, 29.0,43.7, 44.1, 90.6, 100.5, 162.6.

Example 5

This example describes the preparation of 2-cyclohexyl-2-imidazoline bis(nitric oxide) adduct.

The starting material for this preparation was produced by the methoddescribed by Neef et al. (J. Org. Chem. 46: 2824-2826 (1981)). Asolution of 2-cyclohexyl-2-imidazoline (5.0 g, 32.8 mmol) in 300 mlacetonitrile was reacted with NO for 78 h. Yield 6.66 g (97%); m.p.158-159° C. (dec.); ¹H NMR (D₂O) δ1.4-1.7 (6H, m), 1.9-2.1 (2H, m),2.5-2.6 (2H, m), 4.0 (4H, S); ¹³C NMR (D₂O) 23.9 (2C), 26.6, 34.3 (2C),47.3 (2C), 73.0, 173.4.

Example 6

This example describes the preparation of tetrahydrozoline bis (nitricoxide) adduct.

A solution of tetrahydrozoline hydrochloride (10.0 g, 42.25 mmol) in9.66 ml of 25% NaOMe in MeOH (42.25 mmol NaOMe) was diluted with 200 mlacetonitrile and the precipitated sodium chloride was removed byfiltration. The resulting solution was treated with NO for 24 h. Yield9.0. g (82%); m.p. 168-169° C. (dec.); ¹H NMR (D₂O) δ1.8-1.9 (2H, m),2.3-2.45 (1H, m), 2.9-3.0 (3H, m), 4.00 (4H, s), 7.15-7.47 (4H, m); ¹³CNMR (D₂O) 20.4, 30.6, 34.7, 47.7 (2C), 76.0, 129.7, 13.0.7, 131.8,133.0, 133.1, 141.5, 173.7. Anal. Calcd. for C₁₃H₁₆N₄O₂: C, 59.99; H,6.20; N, 21.52. Found: C, 60.05; H, 6.14; N, 21.48.

Example 7

This example describes the preparation of idazoxan-bis(nitric oxide)adduct available from Research Biochemicals, Inc. (Natick, Hi.).

A solution of idazoxan hydrochloride (1.00 g, 4.155 mmol) in a mixtureof 0.95 ml 25% NaOMe in MeOH (4.155 mmol NaOMe) and 3 ml MeOH wasdiluted with 40 ml acetonitrile and the precipitated sodium chloride wasremoved by filtration. The resulting solution was treated with NO for 21h. Yield 0.62 g (56%); m.p. 152-154° C. (dec.); ¹H NMR (D₂O) δ4.04 (4H,s), 4.64 (1H, d), 5.13 (1H, d), 7.02-7.22 (4H, m). Anal. Calcd. forC₁₃H₁₆N₄O₂: C, 49.81; H, 4.94; N, 21.12. Found: C, 50.22; H, 4.61; N,20.98.

Example 8

This example describes a general procedure for preparation ofdiazeniumdiolate derivatives of enamines.

Enamines were prepared from an equimolar mixture of an aldehyde andketone and a wide variety of secondary amines via dehydration. Suchmethods are described in Hicknott, Tetrahedron 38: 1975-2050, and3363-3446 (1982); Cook, Enamines: Synthesis, Structure and Reactions,Marcel Dekker, New York (1988); and Szmuszkovicz, “Enamines”, Chapter 4,In advances in Org. Chem, Methods and Results, Wiley Interscience, NewYork (1963). Preferred amines include dimethylamine, diethylamine,piperidine, pyrrolidine, morpholine and N-methyl-aniline.

These compounds were prepared according to the general procedure setforth in Example 1, except that the reactions were cooled when requiredand some gave crystalline products as indicated in the individualdescriptions.

Example 9

This example describes the preparation of cyclohexanone morpholineenamine bis(nitric oxide) adduct.

A solution of the enamine derived from morpholine and cyclohexanone(15.0 g, 89.7 mmole) in 150 ml ethyl ether was cooled in dry ice withoutstirring and reacted with NO for 20 h as it warmed to room temperature.Workup as above produced large clear crystals of product. Yield 8.14 g(40%); m.p. 85-87° C.; ¹H NMR (CD₃CN) δ1.5-2.3 (6H, m), 2.44-2.55 (4H,m), 2.85-2.96 (4H, m), 5.13-5.18 (1H, m), 5.23-5.27 (1H, t), 11.6 (1H,br.s); ¹³C NMR (CD₃CN) 19.2, 24.7, 28.6, 50.6 (2C), 67.1, 67.5 (2C),112.5, 141.3; exact mass calcd. for C₁₀H₁₇N₃O₃ (M⁺) 227.1269, found227.1254. Anal. Calcd. for C₁₀H₁₇N₃O₃: C, 52.85; H, 7.54; N, 18.49.Found: C, 53.32; H, 7.63; N, 18.76.

Example 10

This example describes the preparation of isobutyraldehyde morpholineenamine bis (nitric oxide) adduct.

A solution of the enamine derived from morpholine and isobutyraldehyde(7:0 g, 49.6 mmole) in 100 ml THF was reacted with NO for 22 h asdescribed above. Yield 4.05 g (41%); m.p. 91-92° C.; ¹H NMR (D₂O) δ1.48(6H, s), 3.25-3.31 (4H, m), 3.92-3.98 (4H, m), 5.26 (1H, s); ¹³C NMR(D₂O) 23.2 (2C), 46.1 (2C), 66.6 (2C), 75.7, 95.2; exact mass calcd. forC₈H₁₆N₃O₃ (MH⁺) 202.1192; found 202.1137. Anal. Calcd. for C₈H₁₅N₃O₃: C,47.75; H. 7.51; N, 20.88. Found: C, 47.74; H, 7.70; N, 20.13.

Example 11

This example describes the preparation of cyclohexanecarboxaldehydemorpholine enamine bis (nitric oxide) adduct.

A solution of 4-(cyclohexylidenemethyl)morpholine (10.0 g, 55.2 mmol) in20 mL of CH₃CN was cooled at 0° C. in an ice bath and reacted withouttirring with NO as described above for 6 h and then warmed to roomtemperature. The product was isolated by filtration, washed with CH₃CN,then ether and dried in vacuo. Yield 7.13 g (54%); mp 115-117° C.; ¹HNMR δ1.25-1.40 (2H, m), 1.48-1.70 (4H, m), 1.95-2.40 (4H, m), 3.20-3.26(4H, m), 3.90-3.96 (4H, m), 5.05 (1H, s); ¹³C NMR δ24.1 (2C), 27.8, 31.3(2C), 46.3 (2C), 67.1 (2C), 78.0, 95.7.

Anal. Calcd for C₁₁H₁₉N₃O₂: C, 54.76; H, 7.94; N, 17.41. Found: C,54.93; H, 8.04; N, 17.60.

Example 12

This example describes the preparation of isobutyraldehyde piperidineenamine bis(nitric oxide) adduct (25).

A solution of the enamine derived from piperidine and isobutyraldehyde(5.0 g, 35.9 mmole) in 150 ml CH₃CN was stirred at room temperature andreacted with NO for 23 h as described above. Yield 3.25 g (45%); m.p.84-85° C.; ¹H NMR (D₂O) δ1.48 (6H, s), 1.66-1.83 (6H, m), 3.13-3.18 (4H,m), 5.25 (1H, s). ¹³C NMR (D₂O) 23.2 (2C), 24.3, 25.1 (2C), 47.4 (2C),75.5, 95.2. Anal. Calcd. for C₉H₁₇N₃O₂: C, 54.25; H, 8.60; N, 21.09.Found: C, 52.69; H, 8.56; N, 21.28.

Example 13

This example describes the preparation of isobutyraldehyde pyrrolidineenamine bis (nitric oxide) adduct.

A solution of N-(2-methyl-1-propenyl)pyrrolidine (10.0 g, 79.9 mmol) in200 mL of CH₃CN was cooled to 0° C. in an ice bath and reacted withoutstirring with NO as described above for 6 h and then warmed to roomtemperature. The product was isolated by filtration, washed with CH₃CN,then ether and dried in vacuo. Yield 88.8 g (60%); mp 75-76° C.; ¹H NMRδ1.48 (6H, s), 1.98-2.03 (4H, m), 3.23-3.32 (4H, m), 5.25 (1H, s); ¹³CNMR δ23.2 (2C), 26.5 (2C), 48.3 (2C), 75.6, 95.2.

Example 14

This example describes the preparation of isobutyraldehydeN-methylaniline enamine bis (nitric oxide) adduct.

A solution of the enamine derived from N-methylaniline andisobutyraldehyde (5.0 g, 31.0 mmole) in 150 ml CH₃CN was stirred at roomtemperature and reacted with No for 20 h. The resulting pale yellowsolution was concentrated to dryness on a rotary evaporator and theresidual solid was recrystallized from absolute ethanol to yield 2.26 g(33% k) of product as pale, cream-colored needles. m.p. 83-84° C.; ¹HNMR (CDCl₃) δ1.59 (3H, s), 1.63 (3H, s), 2.75 (3H, s), 6.00 (1H, s),6.96-7.37 (5H, m); 13C NMR (CDCl₃) 17.4, 26.8, 34.4, 75.6, 101.1, 118.9(2C), 122.7, 129.4 (2C), 149.3. Anal. Calcd. for C₁₁H₁₅N₃O₂: C, 59.71;H, 6.83; N, 18.99. Found: C, 59.77; H, 6.84; N, 19.01.

Example 15

This example describes the preparation of isobutyraldehydeN-methyl-p-toluidine enamine bis(nitric oxide) adduct.

A solution of the enamine derived from N-methyl-p-toluidine andisobutyraldehyde (5.0 g, 28.5 mmole) in 150 ml CH₃CN was stirred at roomtemperature and reacted with NO for 20 h. The resulting paleyellow-orange solution was concentrated to dryness on a rotaryevaporator and the residual off-white solid was recrystallized fromabsolute ethanol to yield 2.21 g (33%) of product as white cotton-likeneedles. m.p. 127-128° C.; ¹H NMR (CDCl₃) δ1.58 (3H, s), 1.61 (3H, s),2.31 (3H, s), 2.71 (3H, s), 5.92 (1H, s), 6.90-7.15 (4H, m); ¹³C NMR(CDCl₃) 17.3, 20.5, 26.8, 34.9, 75.4, 101.9, 119.7 (2C), 129.9 (2C),132.7, 147.2. Anal. Calcd. for C₁₂H₁₇N₃O₂: C, 61.26; H, 7.28; N, 17.86.Found: C, 61.32; H, 7.35; N, 17.88.

Example 16

This example describes the preparation of isobutyraldehydeN-methyl-p-anisidine enamine bis (nitric oxide) adduct.

A solution of the enamine derived from N-methyl-p-anisidine andisobutyraldehyde (5.0 g, 26.1 mmole) in 150 ml CH₃CN was stirred at roomtemperature and reacted with NO for 23 h. The resulting pale brownsolution was concentrated to dryness on a rotary evaporator and theresidual oil was crystallized from absolute ethanol to yield 4.89 g(75%) of product as colorless chunky crystals. m.p. 97-98° C.; ¹H NMR(CDCl₃) δ1.58 (3H, s), 1.60 (3H, s), 2.67 (3H, s), 3.79 (3H, s), 5.80(1H, s), 6.84-7.06 (4H, m); ¹³C NMR (CDCl₃) 17.2, 26.8, 36.1, 55.5,75.2, 103.0, 114.6 (2C), 122.8 (2C), 143.4, 156.3. Anal. Calcd. forC₁₂H₁₇N₃O₃: C, 57.35; H, 6.82; N, 16.72. Found: C, 57.36; H, 6.87; N,16.75.

Example 17

This example describes the measurement of the production of NO and N₂Oby amidine/nitric oxide adducts.

As a demonstration of the efficacy of the amidine/nitric oxide adductsdescribed herein as nitric oxide and nitroxyl releasing agents, selectedcompounds were dissolved in either 0.1 N HCl or pH 7.4 buffer and theheadspace was monitored by chemiluminescence (to detect NO) and gaschromatography (to detect N₂O, the dehydrated dimer of HNO). The resultsare shown in Table I.

TABLE I Cmpd of Ratio Yield (in moles per mole cmpd) Ex. No. SolutionN₂O:NO N₂O NO 2 0.1 N HCl 2:1 0.9 0.45 3 pH 7.4 13:1  0.64 0.05 4 pH 7.46:1 0.45 0.08 5 0.1 N HCl — 0.2 N.D.* 6 pH 7.4 — 0.4 N.D.* *Thecompounds of Examples 5 and 6 released NO too slowly for practicalmeasurement by headspace analysis.

Example 18

This example describes the measurement of the time course of NOproduction by amidine and enamine nitric oxide adducts.

To demonstrate the utility of these compounds as long-term nitric oxidereleasing agents, selected compounds were dissolved in phosphate bufferat pH 7.4 and incubated in a 37° C. thermostated water bath. The NOrelease rate was measured periodically by flushing the solution withinert N₂ gas and then sweeping newly generated NO into achemiluminescence detector and integrating the signal produced over thenext 4-7 mins. NO release was measured over a period of two weeks.

None of these compounds released nitric oxide via a single pathway whichproduced a release profile consistent with first order kinetics.Accordingly, the results of each test are summarized here by giving theinitial NO release rate, the rate at one intermediate timepoint and thetotal time of observed NO release for representative examples.

Thus, the compound of Example V (tetrahydrozoline diazeniumdiolate)showed an initial NO release rate of 3.64×10⁻¹¹ moles NO per minute permilligram of dissolved sample which decreased to 2.06×10⁻¹¹ moles NO permin. per mg. after 7 days and continued for several weeks although thelast quantitative measurement showed an NO release rate of 9.00×10⁻¹²moles NO per min. per mg. 15 days after the beginning of the experiment.

Likewise, the compound of Example VI (idazoxan diazeniumdiolate) showedan initial NO release rate of 5.25×10⁻¹¹ moles NO/min./mg. whichgradually increased to 1.41×10⁻¹⁰ moles NO/min./mg. after 4 days andthen gradually decreased, reaching zero (i.e., no more NO was beinggiven off) by day 16.

Among the enamine-derived compounds, the compound of Example VII (thediazeniumdiolate of the morpholine enamine of cyclohexanone) showed aninitial NO release rate of 4.2×10⁻¹¹ mole NO/min./mg. which decreasedwith nearly first order kinetics to 1.8×10⁻¹¹ mole NO/min./mg. after 3days and reached zero by day 7.

The enamine-derived diazeniumdiolate of Example VIII (from themorpholine enamine of isobutyraldehyde) showed an initial NO releaserate of 3.7×10⁻¹¹ mole NO/min./mg. which rapidly decreased to a rate of7.0×10⁻¹² mole NO/min./mg. and then remained at about this level for 4days before slowly declining, reaching zero after 7 days.

All publications cited herein are hereby incorporated by reference tothe same extent as if each publication were individually and,specifically indicated to be incorporated by reference and were setforth in its entirety herein.

While this invention has been described with emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat the preferred embodiments may be varied. It is intended that theinvention may be practiced otherwise than as specifically describedherein. Accordingly, this invention includes all modificationsencompassed within the spirit and scope of the appended claims.

What is claimed is:
 1. A compound selected from the group consisting of:

wherein R¹-R³ are independently hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₃₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain olefinic, an unsubstituted or substituted C₃₋₁₂ branchedchain olefinic, a substituted or unsubstituted C₃₋₈ cycloalkyl, asubstituted or unsubstituted naphthyl, a substituted or unsubstitutedtetrahydronaphthyl, a substituted or unsubstituted octahydronaphthyl,benzyl or substituted benzyl, substituted with up to three substituents,or phenyl or substituted phenyl, substituted with up to threesubstituents; R⁴ and R⁵ are independently chosen from hydrogen, anunsubstituted or substituted C₁₋₁₂ straight chain alkyl, anunsubstituted or substituted C₃₋₁₂ branched chain alkyl, anunsubstituted or substituted C₃₋₁₂ straight chain olefinic, anunsubstituted or substituted C₃₋₁₂ branched chain olefinic, asubstituted or unsubstituted benzyl, an unsubstituted or substitutedphenyl, amino, an unsubstituted or substituted alkylamino, anunsubstituted or substituted arylamino, an unsubstituted or substituteddialkylamino, an unsubstituted or substituted diarylamino,carboxyalkylamino, carboxydialkylamino, unsubstituted or substitutedtolyl, xylyl, anisyl, mesityl, an unsubstituted or substituted acetyl,an unsubstituted or substituted acetoxy, carboxy, an unsubstituted orsubstituted carboxymethyl, an unsubstituted or substituted carboxyethyl,an unsubstituted or substituted alkylcarbonyl, thiol, an unsubstitutedor substituted alkylthio, an unsubstituted or substituted alkoxy,carboxamido, an unsubstituted or substituted alkylcarboxamido, or anunsubstituted or substituted dialkylcarboxamido, an unsubstituted orsubstituted phenoxy, an unsubstituted or substituted benzyloxy,phenylcarbonyl, benzylcarbonyl, an unsubstituted or substitutednitrophenyl, trialkylsilyl or nitro; R⁴ and R⁵ together with the carbonatom to which they are bonded form an unsubstituted or substituted C₃₋₈cycloalkyl, tetrahydronaphthlene or octahydronaphthalene; and whereinthe substituents on the substituted groups are selected from the groupconsisting of alkoxy, acyloxy, hydroxy, halo, benzyl, acetyl, carboxyl,carboxyalkyl, carboxyalkylamido, carboxydialkylamido, alkylcarbonyl,arylamino, diarylamino, cyano, tolyl, xylyl, mesityl, anisyl,pyrrolidinyl, carboxamido, amino, alkylamino, dialkylamino, formyl,dioxane, thiol, alkylthiol, aryl, heteroaryl, phenoxy, benzyloxy,phenylcarbonyl, benzylcarbonyl, nitrophenyl, trialkylsilyl, nitro,sulfonyl, nitrobenzyl, trialkylammonium, alkyl, cycloalkyl,tetrahydrofuranyl, tetrahydropyranyl, piperidinyl and morpholinyl. 2.The compound of claim 1 of

wherein R¹-R³ are independently hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₃₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain olefinic, an unsubstituted or substituted C₃₋₁₂ branchedchain olefinic, a substituted or unsubstituted C₃₋₈ cycloalkyl, asubstituted or unsubstituted naphthyl, a substituted or unsubstitutedtetrahydronaphthyl, a substituted or unsubstituted octahydronaphthyl,benzyl or substituted benzyl, substituted with up to three substituents,or phenyl or substituted phenyl, substituted with up to threesubstituents; R⁴ and R⁵ are independently chosen from hydrogen, anunsubstituted or substituted C₁₋₁₂ straight chain alkyl, anunsubstituted or substituted C₃₋₁₂ branched chain alkyl, anunsubstituted or substituted C₃₋₁₂ straight chain olefinic, anunsubstituted or substituted C₃₋₁₂ branched chain olefinic, asubstituted or unsubstituted benzyl, an unsubstituted or substitutedphenyl, amino, an unsubstituted or substituted alkylamino, anunsubstituted or substituted arylamino, an unsubstituted or substituteddialkylamino, an unsubstituted or substituted diarylamino,carboxyalkylamino, carboxydialkylamino, unsubstituted or substitutedtolyl, xylyl, anisyl, mesityl, an unsubstituted or substituted acetyl,an unsubstituted or substituted acetoxy, carboxy, an unsubstituted orsubstituted carboxymethyl, an unsubstituted or substituted carboxyethyl,an unsubstituted or substituted alkylcarbonyl, thiol, an unsubstitutedor substituted alkylthio, an unsubstituted or substituted alkoxy,carboxamido, an unsubstituted or substituted alkylcarboxamido, or anunsubstituted or substituted dialkylcarboxamido, an unsubstituted orsubstituted phenoxy, an unsubstituted or substituted benzyloxy,phenylcarbonyl, benzylcarbonyl, an unsubstituted or substitutednitrophenyl, trialkylsilyl or nitro; R⁴ and R⁵ together with the carbonatom to which they are bonded form an unsubstituted or substituted C₃₋₈cycloalkyl, or-tetrahydronaphthlene or octahydronaphthalene; and whereinthe substituents on the substituted groups are selected from the groupconsisting of alkoxy, acyloxy, hydroxy, halo, benzyl, acetyl, carboxyl,carboxyalkyl, carboxyalkylamido, carboxydialkylamido, alkylcarbonyl,arylamino, diarylamino, cyano, tolyl, xylyl, mesityl, anisyl,pyrrolidinyl, carboxamido, amino, alkylamino, dialkylamino, formyl,dioxane, thiol, alkylthiol, aryl, heteroaryl, phenoxy, benzyloxy,phenylcarbonyl, benzylcarbonyl, nitrophenyl, trialkylsilyl, nitro,sulfonyl, nitrobenzyl, trialkylammonium, alkyl, cycloalkyl,tetrahydrofuranyl, tetrahydropyranyl, piperidinyl and morpholinyl. 3.The compound of claim 1 of

wherein R¹-R³ are independently hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₃₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain olefinic, an unsubstituted or substituted C₃₋₁₂ branchedchain olefinic, a substituted or unsubstituted C₃₋₈ cycloalkyl, asubstituted or unsubstituted naphthyl, a substituted or unsubstitutedtetrahydronaphthyl, a substituted or unsubstituted octahydronaphthyl,benzyl or substituted benzyl, substituted with up to three substituents,or phenyl or substituted phenyl, substituted with up to threesubstituents; R⁴ is hydrogen, an unsubstituted or substituted C₁₋₁₂straight chain alkyl, an unsubstituted or substituted C₃₋₁₂ branchedchain alkyl, an unsubstituted or substituted C₃₋₁₂ straight chainolefinic, an unsubstituted or substituted C₃₋₁₂ branched chain olefinic,a substituted or unsubstituted benzyl, an unsubstituted or substitutedphenyl, amino, an unsubstituted or substituted alkylamino, anunsubstituted or substituted arylamino, an unsubstituted or substituteddialkylamino, an unsubstituted or substituted diarylamino,carboxyalkylamino, carboxydialkylamino, unsubstituted or substitutedtolyl, xylyl, anisyl, mesityl, an unsubstituted or substituted acetyl,an unsubstituted or substituted acetoxy, carboxy, an unsubstituted orsubstituted carboxymethyl, an unsubstituted or substituted carboxyethyl,an unsubstituted or substituted alkylcarbonyl, thiol, an unsubstitutedor substituted alkylthio, an unsubstituted or substituted alkoxy,carboxamido, an unsubstituted or substituted alkylcarboxamido, or anunsubstituted or substituted dialkylcarboxamido, an unsubstituted orsubstituted phenoxy, an unsubstituted or substituted benzyloxy,phenylcarbonyl, benzylcarbonyl, an unsubstituted or substitutednitrophenyl, trialkylsilyl or nitro; and wherein the substituents on thesubstituted groups are selected from the group consisting of alkoxy,acyloxy, hydroxy, halo, benzyl, acetyl, carboxyl, carboxyalkyl,carboxyalkylamido, carboxydialkylamido, alkylcarbonyl, arylamino,diarylamino, cyano, tolyl, xylyl, mesityl, anisyl, pyrrolidinyl,carboxamido, amino, alkylamino, dialkylamino, formyl, dioxane, thiol,alkylthiol, aryl, heteroaryl, phenoxy, benzyloxy, phenylcarbonyl,benzylcarbonyl, nitrophenyl, trialkylsilyl, nitro, sulfonyl,nitrobenzyl, trialkylammonium, alkyl, cycloalkyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl and morpholinyl.
 4. The compound of claim1 of

wherein R¹-R³ are independently hydrogen, an unsubstituted orsubstituted C₁₋₁₂ straight chain alkyl, an unsubstituted or substitutedC₃₋₁₂ branched chain alkyl, an unsubstituted or substituted C₃₋₁₂straight chain olefinic, an unsubstituted or substituted C₃₋₁₂ branchedchain olefinic, a substituted or unsubstituted C₃₋₈ cycloalkyl, asubstituted or unsubstituted naphthyl, a substituted or unsubstitutedtetrahydronaphthyl, a substituted or unsubstituted octahydronaphthyl,benzyl or substituted benzyl, substituted with up to three substituents,or phenyl or substituted phenyl, substituted with up to threesubstituents; and wherein the substituents on the substituted groups areselected from the group consisting of alkoxy, acyloxy, hydroxy, halo,benzyl, acetyl, carboxyl, carboxyalkyl, carboxyalkylamido,carboxydialkylamido, alkylcarbonyl, arylamino, diarylamino, cyano,tolyl, xylyl, mesityl, anisyl, pyrrolidinyl, carboxamido, amino,alkylamino, dialkylamino, formyl, dioxane, thiol, alkylthiol, aryl,heteroaryl, phenoxy, benzyloxy, phenylcarbonyl, benzylcarbonyl,nitrophenyl, trialkylsilyl, nitro, sulfonyl, nitrobenzyl,trialkylammonium, alkyl, cycloalkyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl and morpholinyl.
 5. The compound of claim1 Formulae I, II or III, wherein the substituents on the substitutedgroups are selected from the group consisting of benzyl, tolyl,carboxyl, carboxyalkyl, dialkylamino, arylamino, and diarylamino.
 6. Thecompound of claim 2, wherein the substituents on the substituted groupsare selected from the group consisting of benzyl, tolyl, carboxyl,carboxyalkyl, dialkylamino, arylamino, and diarylamino.
 7. The compoundof claim 3, wherein the substituents on the substituted groups areselected from the group consisting of benzyl, tolyl, carboxyl,carboxyalkyl, dialkylamino, arylamino, and diarylamino.
 8. The compoundof claim 4, wherein the substituents on the substituted groups areselected from the group consisting of benzyl, tolyl, carboxyl,carboxyalkyl, dialkylamino, arylamino, and diarylamino.
 9. The compoundof claim 4, wherein R¹ and R² are hydrogen and R³ is the entiresubstituent attached to an amine of a compound selected from the groupconsisting of an amino acid, tryptamine, serotonin, histamine,valcyclovir, adenosine, thyroxine, guanine, guanosine, ubenimex,glucosamine, mannosamine, mycosamine, sphingosine, thienamycin,penicillamine and rimantadine.
 10. The compound of claim 9, wherein theamino acid is selected from the group consisting of lysine, tryptophanand hydroxy-tryptophan.
 11. A pharmaceutical composition comprising thecompound of Formulae I, II or III of claim 1 and a pharmaceuticallyacceptable carrier.
 12. A pharmaceutical composition comprising thecompound of Formula I of claim 2 and a pharmaceutically acceptablecarrier.
 13. A pharmaceutical composition comprising the compound ofFormula II of claim 3 and a pharmaceutically acceptable carrier.
 14. Apharmaceutical composition comprising the compound of Formula III ofclaim 4 and a pharmaceutically acceptable carrier.
 15. A method oftreating an animal having a biological disorder treatable with nitricoxide, which method comprises administering to the animal a compound ofclaim 1 in an amount sufficient to treat the biological disorder in theanimal.
 16. A method of preventing a biological disorder in a mammalsusceptible to prevention with nitric oxide, which method comprisesadministering to the mammal a compound of claim 1 in an amountsufficient to prevent the biological disorder in the mammal.